Azapyrimidine analogues: Inhibition of viral DNA synthesis and protein synthesis in SV40 infected BSC-1 cells

Summary The replication of simian virus 40 DNA and protein synthesis in BSC-1 cells was analyzed in vitro after treatment with 5,6-dihydro-5-azacytidine (DH-5-AzaCR) or 5-azacytidine (5-AzaCR). Results demonstrated that after a 3-h treatment period with 100 μg/ml, DH-5-AzaCR exhibited a 77% inhibition of viral DNA synthesis, whereas 5-AzaCR resulted in a 50% inhibition. Stimulation of DNA synthesis occurred when infected cultures were treated with low doses (0.1 to 0.5 μg/ml) of 5-AzaCR for 3h and after 1 and 2 h of treatment with 100 μg/ml of 5-AzaCR; however, stimulation did not occur with DH-5-AzaCR. DNA synthesized in the presence of either drug demonstrated altered conformations when analyzed on agarose gels; however this alteration was negligible after DH-5-AzaCR treatment, but more pronounced in the presence of 5-AzaCR. Restriction enzyme analysis suggests that DH-5-AzaCR may not be a hypomethylating agent as is 5-AzaCR. Inhibition of total protein synthesis (cellular and viral) was essentially complete 6 h after treatment with DH-5-AzaCR, whereas 5-AzaCR completely inhibited protein synthesis after 3 h. These data indicate that 5-AzaCR does not exhibit a direct dose relationship to the inhibition of DNA synthesis whereas DH-5-AzaCR may show some dose relationship, and that DH-5-AzaCR is a more potent inhibitor of DNA synthesis as compared to 5-AzaCR. This work was supported by grant RR08005, National Institutes of Health, Bethesda, MD. Presented in part before the 87th Annual Meeting of the American Society for Microbiology, Atlanta, GA. April 1–6, 1987.     

Introduction of liposome-encapsulated SV40 DNA into cells

DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.     

Chemical synthesis of tridecanucleoside dodecaphosphate sequence of SV40 DNA

The preparation, by the phosphotriester approach, of d[C-T-A-T-T-C-C-A-G-A-A-G-T] from one tetranucleoside triphosphate and three trinucleoside diphosphate blocks is described. The use of the o-dibromomethylbenzoyl (DBMB) protecting group in oligodeoxyribonucleotide synthesis is described for the first time. Internucleotide linkages are protected by o-chlorophenyl groups which are finally removed by treatment with the N1, N1, N3, N3-tetramethylguanidinium salt of syn-4-nitrobenzaldoxime. The first phosphorylation step (leading to phosphodiester intermediates) is carried out by treatment with o-chlorophenyl phosphorodi-(1,2,4-triazolide) followed by treatment with water and triethylamine. 1-Mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) is used as the activating agent in the second phosphorylation step in which 5'-protected mono- and di-nucleotides are condensed with nucleoside building blocks containing unprotected 3'-hydroxy functions.     

DNA synthesis, mitosis and fusion of myocardial cells

Myocardial cells obtained from embryonic chick ventricles have been used to investigate (1) whether differentiated cells can undergo DNA synthesis and mitosis and, (2) whether heart cells when grown in culture can fuse with each other and with chick skeletal myoblasts to form heterokaryon myotubes. Electron microscopic observations have shown that myocardial cells of day 3 and day 20 chick embryos did contain myofibrils with defined sarcomeres; these cells have been observed in mitosis. Cells obtained by tryptic digestion of day 12 chick ventricles when grown in culture continued to replicate their DNA as shown by thymidine-³H radioautography with DNase controls and were observed in all stages of mitosis. Electron microscopy showed that myofibrils were present in some of the cultured cells. Bi-, tri- and tetranucleate cells were observed in the cultures. Thymidine-³H radioautography showed that these cells were formed by karyokinesis without cytokinesis and by the fusion of uninucleate cells. Since the heart cells could fuse with each other, we tested the possibility that they could fuse with skeletal myoblasts to form heterokaryon myotubes. This was accomplished by co-culturing thymidine-³H labeled ventricular cells and unlabeled skeletal myoblasts. Radioautography with DNase controls showed that some of the myotubes consisted of unlabeled skeletal muscle nuclei and labeled heart nuclei in varied proportions. The factors initiating the formation of these heterokaryons have not been elucidated.     

Stability of polyadenylated RNA in differentiating myogenic cells

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half-life of less than 4 h and the other with a half-life of 17--54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady-state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling.     

DNA synthesis and repair in terminally differentiating embryonic lens cells

In this study, we have investigated the correlation between in vitro differentiation and DNA degradation. We have shown a decrease in high molecular weight DNA in 10-day-old embryonic chick lens cells after 1 day in culture. Using X-ray as a tool to check the ability of these cells to repair single-strand breaks in their DNA, we have observed, in addition to spontaneous DNA degradation, an impairement in the repair process.     

SV40LT highly mutates and immortalizes two fibroblast strains from patients with Wilms' tumor.

In order to analyze in detail the process of immortalization of human cells, SV40LT was introduced into two chromosome 11p- fibroblast strains from Wilms' tumor patients. Both fibroblasts, hereafter referred to as CM1 and CM2, displayed the mutant phenotype in the crisis stage of cellular aging. In comparison to a control fibroblast, the density of the CM1 strain was abnormally high while the crisis period of the CM2 strain was abnormally long. The CM1 immortalization was 7 times greater than the control and the CM2 strain had the highest frequency of immortalization, 7 times greater than the CM1. These findings indicate that genes associated with chromosome 11p- may be involved in the immortalization of human cells. During their abnormal crisis periods, the cells derived from the patients with Wilms' tumor showed an extremely high frequency of chromosomal aberrations and mutations (6TGs --> 6TGr). These results indicate that when the growth-arrested cells from Wilms' patients are induced to grow with the introduction of SV40LT at the crisis stage they are highly mutable, resulting in their immortalization in vitro.     

Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.     

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.     

Intermediate in SV40 DNA Chain Growth

PREVIOUS studies of the DNA replication of simian virus 40 (SV40), an oncogenic member of the papoyavirus group, have been concerned with separation and characterization of replicative intermediates^1–4. Circular replicating intermediates have been identified for SV40^1–3, as well as for the similar replication system of polyoma viral DNA5,6. The replicative intermediates of SV40 DNA have been observed by electron microscopy to contain two forks, three branches and no free ends^1–3 as is the case for the circular replicating molecules of polyoma, bacteriophage λ⁷, Escherichia coli⁸ and colicin E1 in mini-cells^9,10. An important property of replicative intermediates of SV40 DNA that has also been observed in replicating molecules of colicin E1¹⁰ is that most molecules contain a superhelical region in the unreplicated portion of the molecule¹.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

5-methyltetrahydrofolate: synthesis and utilization in normal and SV40-transformed BHK-21 cells.

Dodecyl sulfate complexes of two soluble proteins, serum albumin and fumarase, have been “renatured” with large excesses of the nonionic detergent Triton X-100. The resulting complexes, essentially free of dodecyl sulfate, differ in their sedimentation properties relative to the native protein and in their interaction with Triton X-100. Albumin molecules refold to a form binding only very small amounts of Triton and have a sedimentation coefficient similar to that of the non-denatured protein. On the other hand, refolded fumarase molecules have a lower sedimentation coefficient than that of the native enzyme and bind up to 1.06 mg of Triton/mg protein. It is postulated that the fumarase molecule has been turned “inside-out” by the dodecyl sulfate/Triton treatment, and the implications of such large conformational changes for protein transport across membranes are discussed.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.